Detection of Methylation and Translocation Events by Novel Sequencing Technology ABSTRACT The rapid development of DNA sequencing technologies advanced our understanding of the molecular basis of cancer. However, cancer genomics involves very complex molecular rearrangements, such as translocations, requiring the investigation of long DNA molecules with single nucleotide discrimination. Current Next Generation Sequencing (NGS) technologies cannot address such performance requirements and face challenging limitations. The long term goal of this proposal is to develop a rapid and highly accurate DNA sequencing system that has the potential to fully control the speed and orientation at which the DNA passes through an electronic detector (solid state) both in forward (reading) and reverse (proof read) directions. In contrast with other nanopore technologies, our technology decouples the feeding step from the reading step, enabling to sequence full genomes starting with minute quantity of material. Combined, the characteristics have the potential to provide our sequencing technology with the capability to achieve single nucleotide discrimination with an accuracy of 99.9% or higher using long DNA molecules (200kb). Such a system is anticipated to be capable of accurately reading such long fragments of DNA starting from minute quantities of material without the need of amplification. This would provide both practitioners and cancer researchers with a much better tool than the current state of the art sequencing technologies. Indeed we project that our technology will enable the collection of information of genetic and epigenetic markers of cancer both in expressed and non expressed areas of the genome while reaching time and cost performance metrics currently unachieved on the market. The goal of the current Phase 1 (R43) proposal is to demonstrate that by incorporating our proprietary technology into a sequencing system, we will be able to detect important molecular changes that are associated with cancer initiation and proliferation, to include long range methylations and translocations as well as chromoplexy. Such detection will be rendered possible due to the ability of our system to fully control the translocation rate and linearization of the DNA while passing through the sequencing system detector. In Phase II (R44) will focus on the incorporation of an embedded solid state (electronic) nano- electrode detection component to the initial system developed in phase I for single base nucleotide reading. Provided that targeted milestones are achieved, commercial development of a prototype instrument along with associated consumable chips and reagents will be initiated immediately following the completion of phase II. We project that our technology has the potential to sequence a full human genome in less than one hour at the cost of $200 from biopsy material, thus reaching performance metrics currently unavailable on the current market, and providing cancer researchers and medical practitioners with an invaluable affordable tool for the elucidation of molecular mechanisms of cancer and the detection of specific molecular changes which may improve early clinical detection of the disease.